Spatial constraints within the chlamydial host cell inclusion predict interrupted development and persistence.

Hoare A, Timms P, Bavoil PM, Wilson DP - BMC Microbiol. (2008)

Bottom Line:
As the inclusion expands, the contact between each RB and the inclusion membrane decreases, eventually reaching a threshold, beyond which T3S is inactivated upon detachment and this is the signal for RB-to-EB differentiation.We determine relationships between the length of the T3S needle and the RB radius within an inclusion, and between the RB radius and the number of inclusions per host cell to predict whether persistent infection or normal development would occur within a host cell.These results are all testable experimentally and could lead to significantly greater understanding of one of the most crucial steps in chlamydial development.

Affiliation: National Centre in HIV Epidemiology and Clinical Research, Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia. Ahoare@nchecr.unsw.edu.au

ABSTRACT

Background: The chlamydial developmental cycle involves the alternation between the metabolically inert elementary body (EB) and the replicating reticulate body (RB). The triggers that mediate the interchange between these particle types are unknown and yet this is crucial for understanding basic Chlamydia biology.

Presentation of the hypothesis: We have proposed a hypothesis to explain key chlamydial developmental events whereby RBs are replicating strictly whilst in contact with the host cell membrane-derived inclusion via type three secretion (T3S) injectisomes. As the inclusion expands, the contact between each RB and the inclusion membrane decreases, eventually reaching a threshold, beyond which T3S is inactivated upon detachment and this is the signal for RB-to-EB differentiation.

Testing the hypothesis: We explore this hypothesis through the development of a detailed mathematical model. The model uses knowledge and data of the biological system wherever available and simulates the chlamydial developmental cycle under the assumptions of the hypothesis in order to predict various outcomes and implications under a number of scenarios.

Implications of the hypothesis: We show that the concept of in vitro persistent infection is not only consistent with the hypothesis but in fact an implication of it. We show that increasing the RB radius, and/or the maximum length of T3S needles mediating contact between RBs and the inclusion membrane, and/or the number of inclusions per infected cell, will contribute to the development of persistent infection. The RB radius is the most important determinant of whether persistent infection would ensue, and subsequently, the magnitude of the EB yield. We determine relationships between the length of the T3S needle and the RB radius within an inclusion, and between the RB radius and the number of inclusions per host cell to predict whether persistent infection or normal development would occur within a host cell. These results are all testable experimentally and could lead to significantly greater understanding of one of the most crucial steps in chlamydial development.

Figure 3: (a) The maximum number of RBs attained per inclusion versus RB radius size for one inclusion (blue), two inclusions (red), and five inclusions (magenta). The solid section of the curves corresponds to normal development and the dashed section corresponds to interrupted development leading to a persistent mode of growth. (b) The relationship between EB yield and the maximum number of RBs within a single inclusion. Each point represents a different value for the RB radius, with the radius increasing in the direction of the arrows. Parameter values used were: doubling time td = 1.8 hrs, rate of RB detachment k = 1.3 hrs-1, EB radius re = 0.1 μm, rate of RB-to-EB differentiation μ = 0.25 hrs-1, average spacing between T3S needles on the RB surface L = 0.04 μm, length of T3S needles lp = 0.0078 μm, volume of host cell CellVol = 2400 μm3, proportion of cell not available for inclusion growth ε = 0.3, and threshold number of T3S needles per RB Plim = 23.

Mentions:
Multiple inclusions will reduce the space available within the host cell for each inclusion to grow; i.e., they will in effect reduce the size of each inclusion, and this effectively shifts the threshold curve. We expect that persistence is more likely to occur with greater numbers of inclusions. This is exactly what we observe from our model simulations. We explored the effect of the size of the RB radius on the maximum number of RBs produced per inclusion and also varied the number of inclusions. In Figure 3a we present the maximum number of RBs versus RB radius for the cases of 1 inclusion (blue), 2 inclusions (red), and 5 inclusions (magenta). The solid sections of the curves refer to normal development and the dashed sections refer to interrupted development, leading to persistent infection. We note that the profile for the number of chlamydiae produced increases with RB radius until the point of interrupted development and then decreases (Fig. 3a). The increase with larger RBs is due to increased surface area available for contact with the CIM and thus the inclusion must grow larger, accommodating more RBs, before the threshold contact is reached. If normal development is interrupted, lower numbers of larger RBs will result. This is because growth in the persistent mode coincides with spatial limitations in the host cell being reached; fewer numbers of larger bodies can physically fit in the cell. Increasing the number of inclusions per cell reduces the maximal number of RBs per inclusion (as expected), but more importantly interrupted development is observed to occur for smaller RBs (Fig. 3a) leading to their switching to a persistent mode. The curves in Figure 3a were generated from the deterministic model, setting all parameters to the median values over the range. This resulted in persistence with one inclusion once the RB radius reached 0.665 μm (peak number of RBs is 320). However, this critical RB size decreases to 0.645 μm (203, peak number of RBs) with two inclusions and to 0.615 μm with 5 inclusions (111, peak number of RBs). Interestingly, the number of EBs resulting from infection does not mirror the maximal number of RBs but there is a strong non-linear relationship (Fig. 3b). The maximal number of RBs is greatest when normal development is on the verge of interruption; therefore, this situation results in no EBs being produced. There is an optimal RB radius (0.575 μm) for producing the greatest EB yield (Fig. 3b). Lower than the optimal RB radius, RBs start detaching from the CIM faster and thus do not produce as many (intermediate) bodies with the potential of differentiating to EBs. In contrast, RBs with a radius greater than the optimal remain attached to the CIM for longer and the cell will fill up with chlamydiae before all the RBs have differentiated to EBs. Thus, after the optimal RB radius, increasing the radius results in decreased numbers of EBs and increased numbers of RBs until a persistent mode of growth results (number of EBs produced is zero, Fig. 3b). After this point, increasing the RB radius also decreases the number of RBs produced (Fig. 3b).

Figure 3: (a) The maximum number of RBs attained per inclusion versus RB radius size for one inclusion (blue), two inclusions (red), and five inclusions (magenta). The solid section of the curves corresponds to normal development and the dashed section corresponds to interrupted development leading to a persistent mode of growth. (b) The relationship between EB yield and the maximum number of RBs within a single inclusion. Each point represents a different value for the RB radius, with the radius increasing in the direction of the arrows. Parameter values used were: doubling time td = 1.8 hrs, rate of RB detachment k = 1.3 hrs-1, EB radius re = 0.1 μm, rate of RB-to-EB differentiation μ = 0.25 hrs-1, average spacing between T3S needles on the RB surface L = 0.04 μm, length of T3S needles lp = 0.0078 μm, volume of host cell CellVol = 2400 μm3, proportion of cell not available for inclusion growth ε = 0.3, and threshold number of T3S needles per RB Plim = 23.

Mentions:
Multiple inclusions will reduce the space available within the host cell for each inclusion to grow; i.e., they will in effect reduce the size of each inclusion, and this effectively shifts the threshold curve. We expect that persistence is more likely to occur with greater numbers of inclusions. This is exactly what we observe from our model simulations. We explored the effect of the size of the RB radius on the maximum number of RBs produced per inclusion and also varied the number of inclusions. In Figure 3a we present the maximum number of RBs versus RB radius for the cases of 1 inclusion (blue), 2 inclusions (red), and 5 inclusions (magenta). The solid sections of the curves refer to normal development and the dashed sections refer to interrupted development, leading to persistent infection. We note that the profile for the number of chlamydiae produced increases with RB radius until the point of interrupted development and then decreases (Fig. 3a). The increase with larger RBs is due to increased surface area available for contact with the CIM and thus the inclusion must grow larger, accommodating more RBs, before the threshold contact is reached. If normal development is interrupted, lower numbers of larger RBs will result. This is because growth in the persistent mode coincides with spatial limitations in the host cell being reached; fewer numbers of larger bodies can physically fit in the cell. Increasing the number of inclusions per cell reduces the maximal number of RBs per inclusion (as expected), but more importantly interrupted development is observed to occur for smaller RBs (Fig. 3a) leading to their switching to a persistent mode. The curves in Figure 3a were generated from the deterministic model, setting all parameters to the median values over the range. This resulted in persistence with one inclusion once the RB radius reached 0.665 μm (peak number of RBs is 320). However, this critical RB size decreases to 0.645 μm (203, peak number of RBs) with two inclusions and to 0.615 μm with 5 inclusions (111, peak number of RBs). Interestingly, the number of EBs resulting from infection does not mirror the maximal number of RBs but there is a strong non-linear relationship (Fig. 3b). The maximal number of RBs is greatest when normal development is on the verge of interruption; therefore, this situation results in no EBs being produced. There is an optimal RB radius (0.575 μm) for producing the greatest EB yield (Fig. 3b). Lower than the optimal RB radius, RBs start detaching from the CIM faster and thus do not produce as many (intermediate) bodies with the potential of differentiating to EBs. In contrast, RBs with a radius greater than the optimal remain attached to the CIM for longer and the cell will fill up with chlamydiae before all the RBs have differentiated to EBs. Thus, after the optimal RB radius, increasing the radius results in decreased numbers of EBs and increased numbers of RBs until a persistent mode of growth results (number of EBs produced is zero, Fig. 3b). After this point, increasing the RB radius also decreases the number of RBs produced (Fig. 3b).

Bottom Line:
As the inclusion expands, the contact between each RB and the inclusion membrane decreases, eventually reaching a threshold, beyond which T3S is inactivated upon detachment and this is the signal for RB-to-EB differentiation.We determine relationships between the length of the T3S needle and the RB radius within an inclusion, and between the RB radius and the number of inclusions per host cell to predict whether persistent infection or normal development would occur within a host cell.These results are all testable experimentally and could lead to significantly greater understanding of one of the most crucial steps in chlamydial development.

Affiliation:
National Centre in HIV Epidemiology and Clinical Research, Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia. Ahoare@nchecr.unsw.edu.au

ABSTRACT

Background: The chlamydial developmental cycle involves the alternation between the metabolically inert elementary body (EB) and the replicating reticulate body (RB). The triggers that mediate the interchange between these particle types are unknown and yet this is crucial for understanding basic Chlamydia biology.

Presentation of the hypothesis: We have proposed a hypothesis to explain key chlamydial developmental events whereby RBs are replicating strictly whilst in contact with the host cell membrane-derived inclusion via type three secretion (T3S) injectisomes. As the inclusion expands, the contact between each RB and the inclusion membrane decreases, eventually reaching a threshold, beyond which T3S is inactivated upon detachment and this is the signal for RB-to-EB differentiation.

Testing the hypothesis: We explore this hypothesis through the development of a detailed mathematical model. The model uses knowledge and data of the biological system wherever available and simulates the chlamydial developmental cycle under the assumptions of the hypothesis in order to predict various outcomes and implications under a number of scenarios.

Implications of the hypothesis: We show that the concept of in vitro persistent infection is not only consistent with the hypothesis but in fact an implication of it. We show that increasing the RB radius, and/or the maximum length of T3S needles mediating contact between RBs and the inclusion membrane, and/or the number of inclusions per infected cell, will contribute to the development of persistent infection. The RB radius is the most important determinant of whether persistent infection would ensue, and subsequently, the magnitude of the EB yield. We determine relationships between the length of the T3S needle and the RB radius within an inclusion, and between the RB radius and the number of inclusions per host cell to predict whether persistent infection or normal development would occur within a host cell. These results are all testable experimentally and could lead to significantly greater understanding of one of the most crucial steps in chlamydial development.